PMC:3091642 / 34443-36238 JSONTXT

{"project":"2_test","denotations":[{"id":"20663125-17472750-10592612","span":{"begin":609,"end":611},"obj":"17472750"}],"text":"A two color labeling microarray system was used to compare uninfected- and ILTV infected embryonic lung cells at 1, 3, 5, and 7 dpi. Fluorescently labeled complementary RNA (cRNA) probes were generated by using the Two Color Microarray Quick Labeling kit (Agilent Technologies, Palo Alto, CA, USA) and following the manufacturer's instructions. RNA spike-in controls were used to adjust possible dye effects following manufacturer's instructions. The Spike-in controls represent two sets of ten synthesized RNA mixtures derived from the Adenovirus E1A transcriptome with different concentrations in each set [29]. These spike-in sets were mixed with either uninfected control or infected samples and co-hybridized to arrays. Briefly, 2 μg of total RNA were mixed with Spike-ins and converted to cDNA using reverse transcriptase and oligo dT primers in which T7 promoter sequences were added. T7 RNA polymerase was used for the synthesis and labeling of cRNA with either Cy3 dye for the uninfected control or Cy5 dye for the ILTV infected samples. The fluorescently labeled cRNA probes were purified using the Qiagen RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA), and the concentration, fluorescent intensities, and quality of labeled cRNA probes were determined using a Nano-drop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). An equal amount (825 ng) of Cy3 and Cy5 labeled cRNA probes were hybridized on a 4 × 44 K Agilent custom chicken oligo microarray (array ID: 017698). The hybridized slides were washed using a commercial kit package (Agilent Technologies, Palo Alto, CA, USA) and then scanned using a Genepix 4000B scanner (Molecular Devices Corporation, Sunnyvale, CA, USA) with the tolerance of saturation setting of 0.005%. Three biological replicates were conducted."}